Unraveling the secret life of cryptic algal species: evolution, phylogeography and eco-physiology of the red alga, Bostrychia intricata

Molecular techniques have enhanced our ability to unravel the evolutionary history and hidden diversity of species, and to explain how historical events have helped to shape the demography and dispersal of populations. Cryptic species are typically defined as two or more genetically distinct species that are morphologically indistinguishable. The discovery of cryptic diversity has become a challenge for biologists in understanding the species concepts and biodiversity patterns. Many current studies have revealed the existence of cryptic species, but few studies have focused on their ecological and biological aspects. Bostrychia, a filamentous red alga, has long been used as a model system for studies of evolutionary process and biogeographic history. In the Southern Hemisphere, there are four endemic species: B. arbuscula, B. gracilis, B. intricata and B. vaga. Bostrychia intricata is widely distributed in the Southern Hemisphere, whereas the other three species have more restricted distributions. The aim of this study was to reveal the evolutionary history, phylogeographic pattern and eco-physiological trait within B. intricata. Phylogenetic analysis based on combined data (mitochondrial COI, chloroplast rbcL and nuclear 28S) strongly supported the monophyly of the four Southern Hemisphere Bostrychia species, with B. vaga as a sister species to the other three. Multigene phylogeny and COI-based species delimitation revealed cryptic species diversity within B. intricata and B. vaga. Additionally, a COI-based phylogeographic study indicated the existence of three cryptic B. intricata species (N2, N4 and N5) in New Zealand. Population analyses demonstrated that cryptic species N2 populations recently expanded, possibly after the Last Glacial Maximum (LGM), while N4 was more diverse, showing a stable population, which possibly persisted during the LGM. The results suggested that the contrasting pattern in population structure and demographic histories between cryptic species was probably due to the difference in the evolutionary history and survival ability. Growth experiments clearly showed that cryptic species N4 had a significantly higher specific growth rate than other two species, N2 and N5, in different salinities and temperature, suggesting physiologically differentiation between these cryptic species. Additionally, the small-scale distribution of B. intricata at Moa Point, Wellington of three cryptic species showed that N4 was found at the higher tidal position than species N2 and N5. Cryptic species N2 occurred in more wave-exposed areas than other two species. These results suggest spatial niche differences between cryptic species, possibly allowing them to sympatrically co-exist. This study highlights the fact that cryptic species are distinctly different in many biological characteristics, while maintaining \identical morphologies

First record of the cyanobacterial genus Wilmottia (Coleofasciculaceae, Oscillatoriales) from the South Orkney Islands (Antarctica)

Two cyanobacterial morphotypes isolated from Signy Island, South Orkney Islands, maritime Antarctica were characterised using a polyphasic approach combining morphological, cytological and molecular analyses. These analyses showed that the strains grouped with members of the genus Wilmottia. This genus has three species, W. murrayi, W.stricta, and W. koreana. Both morphotypes analysed in this study were placed within the clade of W. murrayi. This clade showed a well-supported separation from Antarctica and New Zealand strains, as well as the strains from other regions. W. murrayi was first described from Antarctica and is now known from several Antarctic regions. Confirmation of the occurrence of W. murrayi at Signy Island significantly extends its known distribution in Antarctica. In addition, a new combination, W. arthurensis, is suggested for Phormidium arthurensis

Strong and widespread cycloheximide resistance in Stichococcus-like eukaryotic algal taxa

This study was initiated following the serendipitous discovery of a unialgal culture of a Stichococcus-like green alga (Chlorophyta) newly isolated from soil collected on Signy Island (maritime Antarctica) in growth medium supplemented with 100 µg/mL cycloheximide (CHX, a widely used antibiotic active against most eukaryotes). In order to test the generality of CHX resistance in taxa originally identified as members of Stichococcus (the detailed taxonomic relationships within this group of algae have been updated since our study took place), six strains were studied: two strains isolated from recent substrate collections from Signy Island (maritime Antarctica) (“Antarctica” 1 and “Antarctica” 2), one isolated from this island about 50 years ago (“Antarctica” 3) and single Arctic (“Arctic”), temperate (“Temperate”) and tropical (“Tropical”) strains. The sensitivity of each strain towards CHX was compared by determining the minimum inhibitory concentration (MIC), and growth rate and lag time when exposed to different CHX concentrations. All strains except “Temperate” were highly resistant to CHX (MIC > 1000 µg/mL), while “Temperate” was resistant to 62.5 µg/mL (a concentration still considerably greater than any previously reported for algae). All highly resistant strains showed no significant differences in growth rate between control and treatment (1000 µg/mL CHX) conditions. Morphological examination suggested that four strains were consistent with the description of the species Stichococcus bacillaris while the remaining two conformed to S. mirabilis. However, based on sequence analyses and the recently available phylogeny, only one strain, “Temperate”, was confirmed to be S. bacillaris, while “Tropical” represents the newly erected genus Tetratostichococcus, “Antarctica 1” Tritostichococcus, and “Antarctica 2”, “Antarctica 3” and “Arctic” Deuterostichococcus. Both phylogenetic and CHX sensitivity analyses suggest that CHX resistance is potentially widespread within this group of algae

Nodosilinea signiensis sp. nov. (Leptolyngbyaceae, Synechococcales), a new terrestrial cyanobacterium isolated from mats collected on Signy Island, South Orkney Islands, Antarctica

Terrestrial cyanobacteria are very diverse and widely distributed in Antarctica, where they can form macroscopically visible biofilms on the surfaces of soils and rocks, and on benthic surfaces in fresh waters. We recently isolated several terrestrial cyanobacteria from soils collected on Signy Island, South Orkney Islands, Antarctica. Among them, we found a novel species of Nodosilinea, named here as Nodosilinea signiensis sp. nov. This new species is morphologically and genetically distinct from other described species. Morphological examination indicated that the new species is differentiated from others in the genus by cell size, cell shape, filament attenuation, sheath morphology and granulation. 16S rDNA phylogenetic analyses clearly confirmed that N. signiensis belongs to the genus Nodosilinea, but that it is genetically distinct from other known species of Nodosilinea. The D1–D1´ helix of the 16S–23S ITS region of the new species was also different from previously described Nodosilinea species. This is the first detailed characterization of a member of the genus Nodosilinea from Antarctica as well as being a newly described species

Aliinostoc bakau sp. nov. (Cyanobacteria, Nostocaceae), a New Microcystin Producer from Mangroves in Malaysia

A new microcystin-producing mangrove cyanobacterium, Aliinostoc bakau sp. nov., was isolated from a tropical mangrove in Penang, Malaysia, and characterized using combined morphological and phylogenetic approaches. Cultures were established in liquid media of different salinities (0, 7, 14, 21, 28, and 35 ppt). Optimal growth observed at both 7 and 14 ppt was consistent with the origin of the strain from an estuarine mangrove environment. Phylogenetic analysis based on the 16S rRNA gene strongly indicated that the strain is a member of the genus Aliinostoc and is distinct from other currently sequenced species in the genus. The sequences and secondary structure of the 16S–23S ITS region D1–D1’ and Box–B helices provided further confirmation that the new species is clearly distinct from previously described Aliinostoc species. Amplification of the mcyE gene fragment associated with the production of microcystin in A. bakau revealed that it is identical to that in other known microcystin-producing cyanobacteria. Analysis of the extracts obtained from this strain by HPLC-MS/MS confirmed the presence of microcystin variants (MC-LR and -YR) at concentrations of 0.60 μg/L and MC-RR at a concentration of 0.30 μg/L. This is the first record of microcystin production from Aliinostoc species in tropical mangrove habitats

Characterization of five complete Cyrtodactylus mitogenome structures reveals low structural diversity and conservation of repeated sequences in the lineage

Mitochondrial genomes (mitogenomes) of five Cyrtodactylus were determined. Their compositions and structures were similar to most of the available gecko lizard mitogenomes as 13 protein-coding, two rRNA and 22 tRNA genes. The non-coding control region (CR) of almost all Cyrtodactylus mitogenome structures contained a repeated sequence named the 75-bp box family, except for C. auribalteatus which contained the 225-bp box. Sequence similarities indicated that the 225-bp box resulted from the duplication event of 75-bp boxes, followed by homogenization and fixation in C. auribalteatus. The 75-bp box family was found in most gecko lizards with high conservation (55–75% similarities) and could form secondary structures, suggesting that this repeated sequence family played an important role under selective pressure and might involve mitogenome replication and the likelihood of rearrangements in CR. The 75-bp box family was acquired in the common ancestral genome of the gecko lizard, evolving gradually through each lineage by independent nucleotide mutation. Comparison of gecko lizard mitogenomes revealed low structural diversity with at least six types of mitochondrial gene rearrangements. Cyrtodactylus mitogenome structure showed the same gene rearrangement as found in most gecko lizards. Advanced mitogenome information will enable a better understanding of structure evolution mechanisms

Chromosome map of the Siamese cobra: did partial synteny of sex chromosomes in the amniote represent “a hypothetical ancestral super-sex chromosome” or random distribution?

Background Unlike the chromosome constitution of most snakes (2n=36), the cobra karyotype shows a diploid chromosome number of 38 with a highly heterochromatic W chromosome and a large morphologically different chromosome 2. To investigate the process of sex chromosome differentiation and evolution between cobras, most snakes, and other amniotes, we constructed a chromosome map of the Siamese cobra (Naja kaouthia) with 43 bacterial artificial chromosomes (BACs) derived from the chicken and zebra finch libraries using the fluorescence in situ hybridization (FISH) technique, and compared it with those of the chicken, the zebra finch, and other amniotes. Results We produced a detailed chromosome map of the Siamese cobra genome, focusing on chromosome 2 and sex chromosomes. Synteny of the Siamese cobra chromosome 2 (NKA2) and NKAZ were highly conserved among snakes and other squamate reptiles, except for intrachromosomal rearrangements occurring in NKA2. Interestingly, twelve BACs that had partial homology with sex chromosomes of several amniotes were mapped on the heterochromatic NKAW as hybridization signals such as repeat sequences. Sequence analysis showed that most of these BACs contained high proportions of transposable elements. In addition, hybridization signals of telomeric repeat (TTAGGG)n and six microsatellite repeat motifs ((AAGG)8, (AGAT)8, (AAAC)8, (ACAG)8, (AATC)8, and (AAAAT)6) were observed on NKAW, and most of these were also found on other amniote sex chromosomes. Conclusions The frequent amplification of repeats might involve heterochromatinization and promote sex chromosome differentiation in the Siamese cobra W sex chromosome. Repeat sequences are also shared among amniote sex chromosomes, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial syntenies. Alternatively, amplification of microsatellite repeat motifs could have occurred independently in each lineage, representing convergent sex chromosomal differentiation among amniote sex chromosomes

Unraveling the secret life of cryptic algal species: evolution, phylogeography and eco-physiology of the red alga, Bostrychia intricata

Molecular techniques have enhanced our ability to unravel the evolutionary history and hidden diversity of species, and to explain how historical events have helped to shape the demography and dispersal of populations. Cryptic species are typically defined as two or more genetically distinct species that are morphologically indistinguishable. The discovery of cryptic diversity has become a challenge for biologists in understanding the species concepts and biodiversity patterns. Many current studies have revealed the existence of cryptic species, but few studies have focused on their ecological and biological aspects. Bostrychia, a filamentous red alga, has long been used as a model system for studies of evolutionary process and biogeographic history. In the Southern Hemisphere, there are four endemic species: B. arbuscula, B. gracilis, B. intricata and B. vaga. Bostrychia intricata is widely distributed in the Southern Hemisphere, whereas the other three species have more restricted distributions. The aim of this study was to reveal the evolutionary history, phylogeographic pattern and eco-physiological trait within B. intricata. Phylogenetic analysis based on combined data (mitochondrial COI, chloroplast rbcL and nuclear 28S) strongly supported the monophyly of the four Southern Hemisphere Bostrychia species, with B. vaga as a sister species to the other three. Multigene phylogeny and COI-based species delimitation revealed cryptic species diversity within B. intricata and B. vaga. Additionally, a COI-based phylogeographic study indicated the existence of three cryptic B. intricata species (N2, N4 and N5) in New Zealand. Population analyses demonstrated that cryptic species N2 populations recently expanded, possibly after the Last Glacial Maximum (LGM), while N4 was more diverse, showing a stable population, which possibly persisted during the LGM. The results suggested that the contrasting pattern in population structure and demographic histories between cryptic species was probably due to the difference in the evolutionary history and survival ability. Growth experiments clearly showed that cryptic species N4 had a significantly higher specific growth rate than other two species, N2 and N5, in different salinities and temperature, suggesting physiologically differentiation between these cryptic species. Additionally, the small-scale distribution of B. intricata at Moa Point, Wellington of three cryptic species showed that N4 was found at the higher tidal position than species N2 and N5. Cryptic species N2 occurred in more wave-exposed areas than other two species. These results suggest spatial niche differences between cryptic species, possibly allowing them to sympatrically co-exist. This study highlights the fact that cryptic species are distinctly different in many biological characteristics, while maintaining \identical morphologies

Role of Chromosome Changes in Evolution and Diversity

The karyotypes of most species of crocodilians were studied using conventional and molecular cytogenetics. These provided an important contribution of chromosomal rearrangements for the evolutionary processes of Crocodylia and Sauropsida (birds and reptiles). The karyotypic features of crocodilians contain small diploid chromosome numbers (30~42), with little interspecific variation of the chromosome arm number (fundamental number) among crocodiles (56~60). This suggested that centric fusion and/or fission events occurred in the lineage, leading to crocodilian evolution and diversity. The chromosome numbers of Alligator, Caiman, Melanosuchus, Paleosuchus, Gavialis, Tomistoma, Mecistops, and Osteolaemus were stable within each genus, whereas those of Crocodylus (crocodylians) varied within the taxa. This agreed with molecular phylogeny that suggested a highly recent radiation of Crocodylus species. Karyotype analysis also suggests the direction of molecular phylogenetic placement among Crocodylus species and their migration from the Indo-Pacific to Africa and The New World. Crocodylus species originated from an ancestor in the Indo-Pacific around 9~16 million years ago (MYA) in the mid-Miocene, with a rapid radiation and dispersion into Africa 8~12 MYA. This was followed by a trans-Atlantic dispersion to the New World between 4~8 MYA in the Pliocene. The chromosomes provided a better understanding of crocodilian evolution and diversity, which will be useful for further study of the genome evolution in Crocodylia

Contrasting patterns of population structure and demographic history in cryptic species of Bostrychia Intricata (Rhodomelaceae, Rhodophyta) from New Zealand

Spatial patterns of genetic diversity provide insight into the demography and history of species. Morphologically similar but genetically distinct "cryptic" species are increasingly being recognized in marine organisms through molecular analyses. Such species are, on closer inspection, often discovered to display contrasting life histories or occasionally minor morphological differences; molecular tools can thus be useful indicators of diversity. Bostrychia intricata, a marine red alga, is widely distributed throughout the Southern Hemisphere and comprises many cryptic species. We used mitochondrial cytochrome c oxidase I gene sequences to assess the genetic variation, population genetic structure, and demographic history of B. intricata in New Zealand. Our results supported the existence of three cryptic species of B. intricata (N2, N4, and N5) in New Zealand. Cryptic species N4, which was found throughout New Zealand, showed a higher genetic diversity and wider distribution than the other two species, which were only found in the North Island and northern South Island. Our analyses showed low to moderate genetic differentiation among eastern North Island populations for cryptic species N2, but high differentiation among North and South Island populations for N4, suggesting different population structure between these cryptic species. Data also indicated that N2 has recently undergone population expansion, probably since the Last Glacial Maximum (LGM), while the higher genetic diversity in N4 populations suggests persistence in situ through the LGM. The contrasting population structures and inferred demographic histories of these species highlight that life history can vary greatly even among morphologically indistinguishable taxa